Procedure for the counting, detection and identification of mycoplasms in general and urinogenital mycoplasms in particular and a biological medium specially adapted to this effect

ABSTRACT

This process is essentially characterized by enzymatic reactions which are carried out under anaerobic conditions between a liquid growth medium for mycoplasms containing a dilution medium of the sample of fluid to be analyzed and, on the one hand, a first substrate comprising dehydrated urea or glucose in the presence of a color pH indicator also in dehydrated form and, on the other hand, a second substrate comprising arginine also in dehydrated form or glucose in the presence of a color pH indicator and that the speed of the enzymatic response is followed while noting the time corresponding to the color change of the indicators, the respective quantities of urea and arginine or of glucose, on the one hand, and the concentration and the nutrient composition of the said growth and dilution medium, on the other hand, being first selected and standardized in such a way that for Ureaplasma urealyticum present at a supra or sub-pathological rate, the color change of the indicator is or is not obtained after a given amount of time has lapsed.

FIELD OF THE INVENTION

The present invention relates to a procedure for the counting, detectionand identification of mycoplasms in general and urinogenital mycoplasmsin particular. An object of the invention is also a biological mediumspecially adapted to this effect.

BACKGROUND OF THE INVENTION

It is known that mycoplasms are bacteria without walls endowed withenzymatic properties (urease for Ureaplasma urealyticum and argininedecarboxylase for Mycoplasma hominis and fermantans).

These enzymatic properties are used for the identification and countingof the strains in the urinogenital samples.

In effect, these micro-organisms are commensal bacteria (present onperfectly healthy individuals' mucous membrane at a rate slightly loweror equal to 10³ CCU/ml--unit of colour change/ml--). When an infectionbreaks out or when a mucous membrane is rendered fragile (viralinfection, bacteria, hormonal imbalance, etc.), they can proliferate andlead to states of chronic superinfection which can result:

either in tubular sterility;

or in masculine sterility;

or in acute or chronic salpingitis;

or in uretroprostatis syndrome;

or in endocervical dysplasia

(when they superinfect viral infections: Papilloma virus, herpes, CMV,etc.).

In all the cases in question, they are present at a supra-pathologicalrate higher or equal to 10⁴ CCU/ml.

Until now two techniques were used for the counting of urinogenitalmycoplasms:

1. The number of colonies per microscopic field were counted on anisolating gelose.

This technique can be compared to that used for the counting of bacteriain urinary infections (Kass).

However, this type of counting has the inconvenience of systematicallyusing a solid gelose, which is expensive, and is not well adapted forthe systematic research of urinogenital mycoplasms. Furthermore, thetechnique necessitates an incubation in an anaerobic jar.

2. A counting based on the dilution in a series of the sample to beanalysed in a liquid medium (U₉ for U. urealyticum, M₄ 2 for M. hominis)and on the end-point of the appropriate colour indicator of the phenolred type contained in the dilution medium.

However, this technique using the enzymatic properties of urinogenitalmycoplasms has several inconveniences:

the urea, in complex liquid medium, is unstable. Because of this itsconcentration (representing the enzymatic substrate) can vary from oneseries of tests to another, inducing the risk of a lack ofreproductibility;

a reading is not possible until after the colour changes of theindicator have stabilised for at least 24 hours, which involves aminimum response waiting period of 72 hours;

the technique of dilutions in a series is cumbersome and furthermore ismarred by a margin of error due to the manipulation;

no commercial kit exists containing all of the necessary reagents aswell as other materials.

SUMMARY OF THE INVENTION

The present invention proposes to avoid these inconveniences andprovides a process for the counting and detection of mycoplasms usingenzymatic kinetics.

Although this last technique, in effect, is known to be used inbiochemistry to assay enzyme activity, its use has never been suggestedor envisaged to be used for the assay of bacteria, as is the case in theprocess of the present invention.

The Applicant has found, in effect, that the speed of the enzymaticresponse was proportional to the quantity of mycoplasms present in thesample to be analysed.

This process is essentially characterized by the fact that the enzymaticreactions are carried out under anaerobic conditions between a liquidgrowth medium for mycoplasms serving as a dilution medium of the sampleof fluid to be analysed, a first substrate comprising dehydrated urea inthe presence of a pH colour indicator also in dehydrated form and asecond substrate comprising arginine in dehydrated form in the presenceof a pH colour indicator. The speed of the enzymatic response isfollowed by noting the corresponding time for the colour-change of theindicators, the respective quantities of urea and arginine, on the onehand, and the concentration and the nutritious components of the growthand dilution media, on the other hand, being first selected andstandardised in such a way that for the Ureaplasma urealyticum, presentat a supra- or subpathological rate, the turning-point of the colourindicator is or is not obtained after a certain amount of time haslapsed.

According to other features:

the pH indicator is selected with a colour change between pH 6.4 and pH8;

the pH indicator of choice is phenol red;

the concentration of dehydrated urea and of pH indicator, theconcentration of the growth and dilution liquid medium and the quantityof the fluid to be analysed in this medium, are selected in such a waythat at a supra-pathological rate (higher or equal to 10⁴ CCU/ml) ofUreaplasma urealyticum, the indicator turning-point occurs after a timelapse of 24 hours. At a sub-pathological rate of U. urealyticum (loweror equal to 10³ CCU/ml), the indicator change occurs after a time lapseof 48 hours;

the concentration of dehydrated arginine and of the pH indicator, theconcentration of the growth and dilution liquid medium, and the quantityof fluid to be analysed in this medium, are selected in such a way thatthe indicator turning-point occurs in 24 or 48 hours for thesuprapathological rate (higher or equal to 10³ CCU/ml) of M. hominis andfermantans with no turning-point in 48 hours for the sub-pathologicalrate (lower or equal to 10³ CCU/ml);

the dilution medium of the sample to be analysed is essentially based ona growth broth for mycoplasms added with the appropriate nutritiouselements;

the dilution medium for the sample to be analysed advantageouslycomprises a mycoplasm broth, colt serum, ampicillin, an antibiotic, areducing agent of the sodium thioglycolate type, yeast and cysteine.

The process according to the invention is preferably carried out insmall-sized wells, some of which contain dehydrated urea and some ofwhich contain dehydrated arginine and, after the liquid for analysis hasbeen introduced into them, anaerobic conditions are produced by coveringeach well with an inert oily substance such as paraffin oil.

The selection of the concentrations and the standardisation of thereagents for putting the process of the invention into operation aremade by drawing up a calibration curve starting from the countingsaccording to the two usual techniques mentioned above.

More particularly, the object of the invention is also a unique mediumwhich allows the process in question to be put into operation, in asimple manner, requiring only a limited number of manipulations, whilecorresponding to three criteria:

to be stable and able to be stored in a laboratory as well astransported;

to allow the mycoplasms to keep their viability while waiting to be putin their growth medium;

not to influence the quantitative counting of the results.

To reconcile these three necessities, a lyophilisate regeneration mediumis intended to be used in the present invention as a transport mediumfor the sample to be analysed. In effect, mycoplasms are bacteriawithout walls; they are very sensitive to variations in osmotic pressureand have to be introduced quickly into a growth or conservation medium.The disadvantage of growth media is their poor stability, which requiresvery strict stock management by the users in the sample centres. Itshould be recalled that the inconvenience of transport media is thedilution effect when the sample is introduced into them before it is putin the growth medium; this totally alters the quantitative countingwhich is required for calculation of the result.

By unique medium, in the present application, a reactive medium actuallyin the lyophilised form is understood. This medium is alreadyregenerated or is to be regenerated with a dilution medium. Saidreactive medium stabilised in the presence of its diluent is equallyunderstood.

Thus, the unique medium according to the invention comprises

a first so-called reactive phase made up of:

known elements necessary for mycoplasm culture of the cholesterol type,yeast extracts, colt serum,

visualization elements of the mycoplasm metabolism of the urea, arginineor glucose type,

a colour indicator (preferably phenol red) and

at least one antibiotic;

and a second phase, comprising a dilution medium also serving as avehicle for the sample to be analysed, this medium essentiallycomprising a known specific broth of mycoplasms with 1 g/1000 ofagar-agar added and optionally containing an antibiotic.

According to other features:

the antibiotic or antibiotics present in the first phase is (or are)also in the second dilution phase;

this antibiotic(s) is selected from antibiotics of the ampicillin,trimethoprim and nystatin type and preferably from antibiotics acting onthe synthesis of folic acid; these are totally ineffective onmycoplasms;

the dilution medium is free from all unstable elements, so making itsuitable as a transport and conservation medium of a sample given to beanalysed at +4° C.

DETAILED DESCRIPTION OF THE INVENTION

Other features and advantages of the invention emerge more clearly inthe following description and example:

According to the general operational mode of the process of theinvention, at least two wells of a type known per se having a diameterof 9 mm and a depth of 6 mm are used.

In one of these wells, a quantity of 1 mg (milligram) of dehydrated ureawas introduced and in the other a quantity of 1.68 mg (milligram) ofdehydrated arginine was introduced. In each of these wells, 6.8 μg(microgram) of the same dehydrated pH indicator, known as phenol red,was also introduced.

The sample taken from a sick person for analysis purposes was dissolvedusing the medium indicated below (medium A3) corresponding to thefollowing composition and obtained starting from a dehydrated mycoplasmbroth (25.5 g) dissolved in 713 ml of distilled water. The resultingliquid medium had the following added:

200 ml of colt serum;

0.5 g of ampicillin;

8 g of Bactrim® (antibiotic) from Hoffman Laroche, comprising sufamoxoleand trimethoprim;

0.5 g of reducing agent (sodium thioglycolate);

100 ml of yeast extract;

2.5 ml of cysteine (4%).

Each of the wells was covered by a few drops of paraffin oil to createthe anaerobic conditions necessary for the enzymatic reactions.

This composition and the quantities of urea, arginine and pH indicatorwereadapted by carrying out comparative tests using the two traditionaltechniques currently used, in such a way as to obtain the colour changein24 hours for the supra-pathological rate for U. urealyticum and thecolour change in 24 or 48 hours for the suprapathological rate for M.hominis.

For these comparative tests, in the said technique of dilution, thetraditional liquid dilution medium based on urea and arginine was used,ata rate of 150 μl of medium, into which 15 μl of the medium A3 culturedwith the sample taken was introduced, with dilution in series at a rateof 15 μl for each test taken.

For the counting technique per plate (gelose A7), one proceeded asusual, in the evaluation of the mean numbers of colonies of mycoplasmsin the microscope field (at a magnification of 100), in the followingmanner, CFU/ml signifying "colony forming unit"

    ______________________________________                                        less than 1 colony:                                                                             concentration of bacteria                                                     ≦10.sup.3 CFU/ml                                     from 1 to 5 colonies:                                                                           close to 10.sup.4 CFU/ml                                    from 6 to 10 colonies:                                                                          close to 10.sup.5 CFU/ml                                    from 11 to 20 colonies:                                                                         close to 10.sup.6 CFU/ml                                    over 20 colonies: >10.sup.6 CFU/ml                                            ______________________________________                                    

By proceeding in this way starting with urinogenital samples taken frommale or female patients, the correspondence between the differenttechniques was established. This is shown in the following table.

    ______________________________________                                               COUNTING MEDIUM A7                                                     PATIENT  IN LIQUID  No of    count-                                           ARG WELL MEDIUM     colonies ing   UREA WELL                                  ______________________________________                                        A        10.sup.6 in 96 h                                                                         >20      10.sup.6                                                                            Positive 24 h                              B        10.sup.3 in 72 h                                                                          <1      ≦10.sup.3                                                                    Positive 48 h                              C        0 in 96 hours                                                                              0       0.sup.                                                                             --                                         D        10.sup.6 in 72 h                                                                          12      10.sup.6                                                                            Positive 24 h                              E        0 in 96 h    0       0.sup.                                                                             --                                         F        10.sup.1 in 72 h                                                                           0       0.sup.                                                                             --                                         G        contamination                                                                             10 U    10.sup.5                                                                            Positive 24 h                              Positive 24 h   >20 M    10.sup.6                                                                              (unclear)                                    (unclear)                                                                     H        10.sup.4 in 72 h                                                                           3      10.sup.4                                                                            Positive 24 h                              I        10.sup.5 in 48 h                                                                           7      10.sup.5                                                                            Positive 24 h                              ______________________________________                                    

Reference is also made to the curve shown in the appendix.

On examining this table and the attached comparative curve, it can benotedthat for U. urealyticum present at a higher or equal rate to 10⁴CCU/ml, the well with urea changes colour (indicator turning-point) in24 hours. The colour change of this same well in 48 hours correspondseither to the presence of U. urealyticum at a lower or equal rate to 10³CCU/ml (without significance in human pathology), or to chemical orbacterial interference. The urea wells have a great sensitivity: nocolourchange in 48 hours in the urea well confirms the total absence ofU. urealyticum even at a low rate (10² to 10³ CCU/ml) the greatsensitivity of the urea wells being given.

Furthermore, it can be noted that no interference (bacterial orchemical) can alter the response in 24 hours, resulting in a perfectreliability of detection which can always be verified by transferringthe A3 medium on a solid gelose A7 (while using the wells for ascreening).

For M. hominis and fermantans, the enzymatic activity being weaker, onlya colour change in the arginine well (ARG) in 48 hours allows thedetection of a sub-pathological rate (lower or equal to 10³ CCU/ml withno colour change in the well in 48 hours) and supra-pathological rate(higheror equal to 10⁴ CCU/ml with a colour change in the well in 24 or48 hours).

It follows that the colour change of the urea well in 24 hours and inthe arginine well in 24 hours or 48 hours are interesting. Themicro-biologistcan therefore, in this time lapse, confirm whether or notthere is an infection or superinfection of a mucous membrane by amycoplasm.

In reference to the above table, it is noted that for patient F, theresults correspond to a concentration which does not seem to be detectedby the present technique. This is due to the fact that the threshold ofsensitivity of the wells was voluntarily fixed at 10³ CCU/ml. The lowerconcentrations do not have any interest in the diagnosis of mycoplasminfections. It is normal not to find colonies on the medium A7, becausethe technique of direct inoculation induces a dilution factor.

As for patient G, it is noted that only the counting per plate can beinterpreted.

It is noted that the countings carried out by means of the threetechniquesin question lie in a straight correspondence line between thegelose A7 technique and the dilution technique and are situated on thechanging kinetic curve corresponding to it (24 hours for the higher orequal concentrations at 10⁴ CCU/ml and 48 hours for the lowerconcentrations at 10³ CCU/ml).

As indicated previously, it is noted on the attached graph, that onlypatient F does not lie on this straight line.

It is noted that for the supra-pathological concentration, the timelimit for obtaining values by the dilution technique is 72 hours, 48hours for the counting plates, 24 hours with the counting in wells,according to thepresent invention.

Moreover, the Applicant notes that the new technique according to theinvention and using enzyme kinetics on the supports containing thedehydrated substrate, has a very good correlation in 95% of the caseswhencompared to the two other techniques used.

The 5% margin of error, noted in the course of several experiments canbe attributed:

either to strains which preferentially grow in liquid media, as is thecasewhen the counting on solid gelose is wrongly used;

or with strains which have a weak enzymatic activity, as is the casewhere the only countings on plates gives low values.

Therefore, it can be observed that the detection of this 5% margin oferrorcan be made by simultaneously using the two methods of counting,one of thepresent invention and one with solid gelose.

Furthermore, enzyme kinetics in wells according to the presentinvention, in which the substrate exists in the dehydrated form, has,with respect tothe dilution technique in series, the followingadvantages:

stability of the substrate guaranteeing reproductibility;

fewer manipulations and elimination of imprecision resulting from manualdilutions on micro-plates with the small samples used in the assay;

response waiting time of 24 rather than 72 hours;

and finally a cost price reduction when all the materials used are takeninto account.

A particular operational mode uses the unique medium describedpreviously in the present invention.

The sample to be analysed from a patient (a urinogenital sample wastaken here as the example) is introduced as an aliquot of the so-calleddilutionmedium and stored in a flask.

If the active phase exists in lyophilised form, the so-called reactivemedium is "regenerated" by the addition of dilution medium. Clearly, ifthe reactive medium is already diluted and stabilised itself, it is usedas it is.

An aliquot of the medium containing the sample is poured into an aliquotamount of the reactive medium. The detection, that is, visualisation ofthe presence of the bacteria under test, is made evident by the colourchange of the indicator turning-point (from yellow to red). Furthermore,the medium remains clear because urinogenital mycoplasms, in contrast toother bacteria, do not cloud the medium.

The counting of mycoplasms whose presence is made evident, is carriedout by the enzyme kinetic procedure described above. This technique isused toadvantage in active media (regenerated), as described in theinvention, to establish a differential diagnosis between the strainspresent at a supra-pathological rate (higher or equal to 10³ CCU/ml:colour changein 24 hours) and those present at a sub-pathological rate(lower or equal to 10² CCU/ml; colour change in 48 hours or more).

In cases where the counting is of less practical interest (search formycoplasms at a very low rate, e.g. in the case of the supervision of invitro fertilisation, examination of sperm, in haemocultures or insurgicalremoval in salpingitis), the observation of the end-point in themedium is made after the first 24 hours.

In all other cases in question, the technique used for the counting, inparticular the technique of enzyme kinetics, directs the practitionertowards the level of mycoplasms present in the sample taken.

For the identification of the mycoplasms it is intended, according tothe invention put forward here, to follow up the operations of detectionand counting carried out with the biological medium, by identificationof the mycoplasms using their sensitivity profile to antibiotics(antibiogram).

In effect, such an antibiogram of the mycoplasms not only establishesthat the colour change of the unique medium shown here is definitely dueto a mycoplasm, and not to other bacteria having identical enzymaticactivity, but also the profile found allows the confirmation of whichtype, species and genus is present. As soon as the change of colour ofthe indicator is noted and the medium is still clear (24 hours forexample for the traditional urinogenital samples taken, urethra, vagina,endocol, and above 24 hours for the other cases cited above), theoperation of this phase of identification is carried out on the samemultiplate well, regrouping the identification characteristics and theantibiotics to be tested with a second aliquot of another reactivemedium identical to the first, to which the previous medium used isadded.

Thus, it can be observed and noted that the profile of this type ofmultiplate well is well known in the technique, which allowsdifferentiation between Ureaplasma and Mycoplasma (hominis orfermantans) and the assessment of the sensitivity of the isolated strainto the antibiotics present (results from 24 to 48 hours).

It can also be seen that with this unique medium, supplemented with aspecific antibiogram multiplate well, it is possible to achieve, in from48 to 72 hours, the transport of a sample, the detection, quantitativeevaluation, identification and sensitivity to antibiotics, moreparticularly 98% of urinogenital mycoplasm strains responsible forinfections (Mycoplasma fermantans represents less than 2% of thispathology; it is however detected, although assimilated with hominis byits arginine-positive character).

The interest of the medium in this invention therefore lies in:

1. The simplicity of the technique.

2. The absence of false positives (Contaminants seldom show theirenzymaticcharacteristics in 24 hours. Furthermore, the semi-gelosemedium allows thepresence of possible contaminants resistant toampicillin, trimethroprim and nystatin; such contaminants cloud themedium.

However, if certain strains sensitive to the antibiotic mixture presentin the medium of the invention (liquid remaining clear) express theirenzymatic urea or arginine-positive character (broth clear red), itwould be impossible to falsely render a test positive, it being giventhat the antibiogram multiplate well would show an aberrant profile andthe possible colour change of the well containing "contaminant" presentin this multiplate well.

3. The possibility of having a stable transport medium (separation ofthe labile factors present in the lyophilisate).

4. The possibility of systematically introducing in the diagnosis of themycoplasms a sensitivity study to the antibiotics. This is all the morejustified in that 15% of the strains are resistant to tetracyclines andmore than 30% of Ureaplasma urealyticum and 97% of Mycoplasma hominisare resistant to erythromycin (Congress I.C.A.A.C. 25 New York, 1987).

5. The possibility of diagnosing in a precise manner 10% of the strainsgrowing preferentially in a liquid medium, which until now wereimpossibleto distinguish from possible contaminants (urea orarginine-positive and sensitive to the antibiotics present in themedium: clear media having changed to red. Example: campylobacter,certain anaerobic or microaerophilic bacteria of the vaginal flora). Asthese 10% of strains donot grow on solid isolating geloses, thediagnosis was always uncertain.

6. The possibility of totally eliminating the anaerobic conditions inanaerobic jars or sachets, which are relatively expensive and sometimestoo sophisticated for non-specialised laboratories.

It goes without saying that the present invention has only beendescribed only in a purely explanatory manner and is no way limiting.All possible useful modifications can be brought about as equivalentswithout exceedingthe scope of the present invention.

It is particularly in this way that the unique medium according to theinvention can also be used for diagnosing pneumonary mycoplasms (anexample of which are atypical pneumopathies) by means of using glucoseinstead of urea and arginine, together with the appropriate indicator(Thymol Blue).

We claim:
 1. A method for the detection and quantitation of mycoplasmascomprising:reacting a sample to be analyzed under anaerobic conditionsin a liquid mycoplasma growth medium to obtain an enzymatic response,said mycoplasma growth medium also being used as dilution medium for thesample to be analyzed, said sample reacted with:(a) a first substrateconsisting essentially of dehydrated urea in the presence of adehydrated colored pH indicator; (b) a second substrate consistingessentially of dehydrated arginine in the presence of a colored pHindicator; and (c) an optional third substrate comprising glucose in thepresence of a dehydrated pH indicator; and measuring the enzymaticresponse by measuring the time required for the pH indicators to changecolor; the respective amounts of urea and arginine and the concentrationand the composition of the growth medium having been chosen andstandardized such that, for U. urealyticum and M. hominis or M.fermentans or pulmonary mycoplasmas present at supra- orsub-pathological levels, any color change of the pH indicators isobtained within a predetermined length of time; whereinsupra-pathological levels of mycoplasmas are levels greater than 10⁴CCU/ml, and sub-pathological levels of mycoplasmas are levels less than10³ CCU/ml.
 2. The method according to claim 1 wherein the pH indicatorchanges color at a pH between 6.4 and
 8. 3. The method according toclaim 2 wherein the pH indicator is selected from the group consistingof Phenol Red and Thymol Blue.
 4. The method according to claim 1wherein the concentration of dehydrated urea and the pH indicator insaid first substrate and the concentration of growth medium and thequantity of fluid sample to be analyzed are such that, atsuprapathological levels of U. urealytium, the color change of the pHindicator occurs within 24 hours and that at subpathological levels ofU. urealyticum the color change of the pH indicator occurs within 48hours.
 5. The method according to claim 1 wherein the concentration fdehydrated arginine and the concentration of the pH indicator in saidsecond substrate and the concentration of growth medium and the quantityof sample to be analyzed are such that, at suprapathological levels ofM. hominis and M. fermentans, the color change of the pH indicatoroccurs within from 24 to 48 hours and that for subpathological levels ofM. hominis and M. fermentans, no color change occurs within 48 hours. 6.The method according to claim 1 wherein the dilution medium for thesample to be analyzed is based upon a mycoplasma growth brothsupplemented with appropriate nutritional elements.
 7. The methodaccording to claim 6 wherein the dilution medium for the sample to beanalyzed comprises mycoplasma broth, foal serum, ampicillin, anantibiotic, a reducing agent of the sodium thioglycolate type, yeastextract and cysteine.
 8. The method according to claim 1 wherein themethod is conducted in wells of small dimensions, a first set of wellscontaining a substance selected from the group consisting of urea andglucose, and a second set of wells containing a substance selected fromthe group consisting of dehydrated arginine and glucose, and said wellsare covered with an inert oil to establish anaerobic conditions.
 9. Themethod according to claim 8 wherein said inert oil is paraffin oil. 10.The method according to claim 1 wherein the concentrations and thereagents are obtained by establishing a correlation curve usingnumerations according to conventional techniques.